An electron mediator is also referred to as an electron transfer substance, meaning a substance that has a function to receive an electron from an electron donor and/or a function to give an electron to an electron acceptor, and it is known that such mediators exist in an oxidized form or a reduced form. The electron mediator is used for colorimetric determination of enzyme activity, etc. and plays a role in giving an electron received from an enzyme through an enzyme electrode.
An enzyme electrode is used for an enzyme sensor, etc. to measure the content of a specific substance (target substance) included in a biological sample by utilizing an enzyme. Various enzyme sensors have already been commercialized, but for example, a glucose sensor, etc. used to measure glucose concentration in the blood is known, and for an electrode, an enzyme electrode in which an enzyme is immobilized on the surface of an electrode such a gold electrode, a platinum electrode, or a carbon electrode is used. In a glucose sensor, a substance generated by a reaction between glucose and an enzyme in a sample is electrochemically detected and quantitated.
As an example of use of an enzyme electrode, a bio-battery utilizing an electron generated by enzyme treatment with glucose or ethanol as a substrate is known. As examples of bio-batteries, self-contained batteries and environment-friendly batteries, etc. have been focused on in recent years.
In general, because an enzyme is not prone to direct oxidation or reduction on an electrode surface, an electron mediator that plays a role in receiving an electron from the enzyme and giving it to the electrode is necessary in order to measure the glucose concentration in a biological sample.
As an electron mediator, a protein electron mediator has been developed, and as an electrode using a protein electron mediator, an enzyme electrode using cytochrome C, cytochrome b562, and cytochrome c551, etc. (Patent Document 1) is known. Furthermore, a fusion protein (Patent Document 2), etc. in which the cytochrome C domain of quinohemoprotein ethanol dehydrogenase derived from Comamonas testosterone, which is a protein electron mediator, is fused with pyrroloquinoline quinone glucose dehydrogenase has been developed. At the same time, glucose sensors using flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase derived from Burkholderia cepacia have been developed (Patent Documents 3 and 4) and an enzyme electrode including an enzyme derived from Burkholderia cepacia and a production method thereof have also been reported (Patent Document 5).
The cytochrome C and cytochrome b562 described in Patent Document 1 are both electron transfer proteins present in a cell such as a cell membrane or periplasm. When this electron transfer protein performs electron reception to an electrode in the absence of other electron mediators, it requires an amount 100 times as much at a molar ratio compared to glucose dehydrogenase, and it can therefore be said that it has very low affinity with glucose dehydrogenase and glucose oxidase. The fusion protein described in Patent Document 2 requires a massive amount of as many as 1,000 units when glucose measurement is performed. In addition, in glucose measurements performed using the fusion protein, the range of measurable glucose concentrations is 5 mM or less, but it can be said that this is very narrow compared to the fact that the upper limit of glucose concentration generally obtained in blood sugar measurements is 20 to 40 mM. The sensors described in Patent Documents 3, 4, and 5 use an enzyme comprising three subunits including an α subunit, in which the original wild-type enzyme is a catalytically active subunit, a β subunit, in which the original wild-type enzyme is an electron transfer subunit, and a γ subunit, and these are sensors in which a trimer of αβγ subunits or a dimmer of αβ subunits are immobilized. Because the enzyme originally has an electron transfer subunit (β subunit) equivalent to cytochrome C as a wild type, it is an enzyme capable of direct electron migration to an electrode. However, because the β subunit is a membrane-bound cytochrome and the enzyme is a membrane-bound enzyme derived from Burkholderia cepacia, complicated treatments such as solbilization treatment are required in order to obtain a subunit of the enzyme or the enzyme. Additionally, because those that have been subjected to solbilization treatment are unstable, it is difficult to maintain the enzyme or its subunit structure once processes such as desiccation is performed. In addition, according to a presentation at the Annual Meeting of the Society for Biotechnology, Japan (Oct. 28 to 30, 2002), the enzyme has poor substrate specificities to maltose activity and galactose activity when the substrate specificity to glucose activity is 100%, the substrate specificities being 40% and 105% for SM4 strains, 43% and 132% for JCM5506 strains, 57% and 123% for JCM550 strains, 83% and 108% for JCM2800 strains, 74% and 117% for JCM2801 strains, 38% and 104% for IFO14595 strains, and 74% and 148% for IFO15124 strains from among each strain of Burkholderia cepacia, and according to the presentation of the presenter, because it has high active properties on maltose and galactose, there are problems for using it for a self-blood glucose meter.
In Patent Documents 3, 4, and 5, a glucose sensor using a FAD-dependent enzyme and electrode are disclosed, but here, an enzyme is immobilized to a powder conductor or a carbon particle that are different from the electrode. Moreover, platinum, etc. is used for a counter electrode, which is a second electrode, and the second electrode does not have redox properties. Furthermore, no polymer molecules are used for supporting an enzyme against an electrode.
As other types of cytochrome, extracellular secretion type cytochrome b562 derived from Phanerochaete chrysosporium is known, and regarding recombinant cytochrome in which the cytochrome has been produced with yeast, it is known that it has sorbability to cellulose and chitin (Non-patent Document 1). However, regarding the cytochrome, its function as a mediator for glucose oxidoreductase was not known. Furthermore, because the recombinant cytochrome has yeast as a host, excessive sugar chains are added, causing a large molecular weight, and therefore, the solid content per mole is high, and for a reagent for a sensor in which it is necessary to dissolve a reagent with a small amount of blood, there has been a need for a recombinant protein with less glycosylation and a smaller molecular weight.